Bacillus anthracis is a member of the B. cereus group that includes B. cereus and B. thuringiensis. Three bacterial species share >90% identical genomes at the nucleotide level, but they are differentiated by host specificity and/or disease manifestation. Whereas B. thuringiensis is mainly an insect pathogen and B. cereus causes a nonlethal food poisoning in humans, B. anthracis infections (anthrax) are extremely lethal in mammals. The virulence of B. anthracis has been primarily attributed to its two plasmids pXO1 and pXO2, which carry the structural genes encoding the anthrax toxins and genes involved in the synthesis of the poly-D- glutamate capsule. These virulence plasmids are absent in typical isolates of B. cereus and B. thuringiensis. However, it is also evident that regulatory mechanisms for virulence gene expression are distinct in B. anthracis. PlcR, for example, has been known to be a master virulence regulator in B. cereus and B. thuringiensis, but is nonfunctional in B. anthracis due to a nonsense mutation in plcR. PlcR constitutes a peptide-based quorum sensing system with PapR, which is produced as a pre-propeptide, secreted and processed proteolytically into an active form of PapR. Upon reaching a sufficient extracellular concentration, the active PapR is imported into the cell, in which it binds and activates PlcR, subsequently controlling expression of target virulence genes. To date, whether a peptide-based quorum sensing system exists in B. anthracis is completely unknown. Results from our preliminary study strongly indicate that B. anthracis possesses an as-yet-uncharacterized peptide-based quorum sensing system, named AqsR and AqsP for Anthrax quorum sensing Regulator and Peptide, which appears to be distinct from PlcR-PapR. Inactivation of AqsR-AqsP renders B. anthracis attenuated in a murine model of pulmonary anthrax, suggesting that the AqsR-AqsP system regulates expression of virulence-related genes in B. anthracis. In proposed studies, we will identify genetic components constituting and controlling the AqsR-AqsP quorum sensing system, and identify and characterize AqsR/AqsP-regulated virulence genes. An accomplishment of these goals will establish the AqsR-AqsP quorum sensing as a new virulence regulatory mechanism in B. anthracis, advance our knowledge about the pathophysiology of B. anthracis, and help the development of better anti-anthrax strategies. Moreover, because an intact AqsR-AqsP appears to be conserved in both B. cereus and B. thuringiensis, knowledge obtained in this study will also be applicable to the physiology of the B. cereus group of bacteria. PUBLIC HEALTH RELEVANCE: Existence of a peptide-based quorum sensing system and its relevance for virulence have not been demonstrated in Bacillus anthracis. Achieving the goals in the proposed research will define the genetic components of a novel quorum sensing system and establish how this regulatory system controls virulence gene expression in B. anthracis. Results from this project will advance our knowledge about B. anthracis pathophysiology and help develop better anti-anthrax strategies.